CN102336813B - A kind of preparation method of synthesizing proteidin with solid phase polypeptide - Google Patents
A kind of preparation method of synthesizing proteidin with solid phase polypeptide Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of synthesizing proteidin with solid phase polypeptide; does technical scheme of the present invention comprise the following steps: with Rink? Amide? mbha resin is starting raw material; HBTU/HOBt is condensing agent; the amino acid with Fmoc-blocking group is connected successively according to the method for solid phase synthesis; last amino acid uses Boc-Arg-OHHCl, obtains 17 peptide resins of protection, then adds and cuts peptide reagent and carry out cutting peptide; add ether sedimentation, obtain reduced form crude product.-SH base the Trt of 5-position and 14-position Cys protects, and is under the condition of 8.5 ~ 10.0 with hydrogen peroxide oxidation at pH;-SH base the Acm of 7-position and 12-position Cys protects, and with iodine oxidation in methanol solution, obtains two disulfide linkage Cys
5-14cys
7-12crude product, finally adopt C8 high performance liquid phase post to carry out separation and purification, obtain target product, purity is 99.21%, and total recovery is: 23.68%.Method of the present invention, production cost is low, and technique is simple, and environmental pollution is low, and productive rate is high, is convenient to industrializing implementation.
Description
Technical field
The present invention relates to a kind of preparation method of proteidin, be specifically related to the Solid phase peptide synthssis preparation method of proteidin.
Background technology
Chinese name: proteidin
English name: Protegrin, Iseganan
Structural formula:
Molecular formula: C
78h
126n
30o
18s
4hCl
Molecular weight: 1900.32+36.46
No. CAS: 256475-21-5
Proteidin is natural existence in mammalian body, is distributed in the First Line of bacterium and fungi invasion, has the activity of broad spectrum antimicrobial, farthest can reduce the possibility that microorganism produces resistance.At I clinical trial phase of the U.S., IntraBiotics drugmaker thinks that it is treatment cystic fibrosis (CF) new drug likely, design experiment formulation is Perorally administrable antimicrobial cellulose solution.This medicine is in third stage experimental stage at present, the infection caused after indication is defined as prevention and treats head or neck cancer radiation treatment or tumour.
The polypeptide compounds that proteidin is made up of ten seven amino acids, is characterized in containing four halfcystines and two pairs of disulfide linkage.At present, also not about the report of the Industrialized processing technique containing two pairs of disulfide linkage polypeptide products, therefore, technique has a great deal of practical meanings.
Summary of the invention
The object of the invention is the preparation method of openly a kind of synthesizing proteidin with solid phase polypeptide, the present invention proposes a more suitable route, use Solid phase synthesis proteidin, and yield is higher.
Technical scheme of the present invention comprises the following steps:
With RinkAmideMBHA resin for starting raw material, with the amino acid of Fmoc protection for monomer, with 23% hexahydropyridine for reagent of raising one's hat, HBTU/HOBt, for connecing peptide condensing agent, connects amino acid successively one by one, last amino acid adopts Boc-Arg-OHHCl, then add and cut peptide reagent and carry out cutting reactive polypeptide, add ether sedimentation, obtain reduced form crude product,-SH base the Trt of 5-position and 14-position Cys protects, and is under the condition of 8.5-10.0 with hydrogen peroxide oxidation at pH;-SH base the Acm of 7-position and 12-position Cys protects, and with iodine oxidation in methanol solution, obtains two disulfide linkage Cys
5-14cys
7-12crude product.Finally adopt HPLC(C8 post) carry out separation and purification, obtain proteidin fine work.
According to the solution of the present invention, with RinkAmideMBHA resin for starting raw material, connect the amino acid with Fmoc-blocking group successively, obtain 17 peptide resins of protection, slough the method for Fmoc-blocking group therebetween successively, comprise the steps:
(1) preparation of Fmoc-Arg (Pbf)-resin
RinkAmideMBHA resin DMF soaks, and makes resin fully swelling, drains, and adds the DMF solution of hexahydropyridine, and 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.Add the Fmoc-Arg (Pbf)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
Said RinkAmideMBHA resin substitution amount is: 0.3-0.6mmol/g;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(2) preparation of Fmoc-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Gly-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(3) preparation of Fmoc-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Val-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(4) preparation of Fmoc-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Cys (Trt)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(5) preparation of Fmoc-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Val-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(6) preparation of Fmoc-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add be dissolved in DMF Fmoc-Cys (Acm)-OH, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(7) preparation of Fmoc-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Phe-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(8) preparation of Fmoc-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Arg (Pbf)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(9) preparation of Fmoc-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Gly-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(10) preparation of Fmoc-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Arg (Pbf)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture is carried out connect peptide 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(11) preparation of Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add be dissolved in DMF Fmoc-Cys (Acm)-OH, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(12) preparation of Fmoc-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Tyr (tBu)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(13) preparation of Fmoc-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Cys (Trt)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(14) preparation of Fmoc-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add Fmoc-Leu-OH, HBTU of being dissolved in DMF, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(15) preparation of Fmoc-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Gly-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(16) preparation of Fmoc-Gly-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Gly-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is carried out connecing reactive polypeptide 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(17) preparation of Boc-Arg-Gly-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Boc-Arg-OHHClH being dissolved in DMF
2o, DIC, HOBT, undertaken connecing reactive polypeptide 12 hours by mixture 20-22 DEG C.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
Three times are washed again with anhydrous methanol; After draining, put into vacuum drier dry, weigh, obtain protection 17 peptide resin;
The DMF strength of solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(18) H-Arg-Gly-Gly-Leu-Cys (H)-Tyr-Cys (Acm)-Arg-Gly-Arg-Phe-Cys (Acm)-Val-Cys (H)-Val-Gly-Arg-NH
2preparation
17 peptide resins of protection are transferred to and are cut in peptide bottle, under cooling, add while stirring and cut peptide reagent, and 15-18 DEG C is continued stirring and carry out cutting reactive polypeptide 4 hours.Filter, drain, filtrate adds anhydrous diethyl ether precipitation, obtains reduced form 17 peptide crude product;
（19）
-NH
2single disulfide linkage Cys
5-14the preparation of crude product
Reduced form 17 peptide crude product is dissolved in purified water, under agitation slowly adds 1mol/L ammoniacal liquor, regulate pH to 8.5 ~ 10.0, add hydrogen peroxide 50ml and stir 30 minutes (HPLC follows the tracks of reaction process), add acetic acid to pH=5 ~ 6, cross and filter insolubles;
Crude product is dissolved in concentration in purified water: 1.5-2.5mg/ml;
By ammoniacal liquor adjustment pH scope be: 8.5 ~ 10.0;
(20)
two disulfide linkage Cys
5-14cys
7-12the preparation of crude product
At single disulfide linkage Cys
5-14in the aqueous solution of crude product, under agitation slowly add the methanol solution of iodine, stir about 60 minutes, HPLC follows the tracks of reaction process, drips 10% hypo solution termination reaction.After decompression removing methyl alcohol, reclaim iodine 4 times, with 0.4 μm of membrane filtration with carbon tetrachloride extraction;
The concentration of methanol solution of iodine is: 126g iodine is dissolved in 1000ml methyl alcohol;
According to the preferred technical scheme of the present invention, separation and purification comprises the steps:
Filtrate is through the separation and purification of C8 filled column, and moving phase is: phosphoric acid triethylamine/acetonitrile; Flow velocity is: 500-600ml/min; Determined wavelength is: 260-280nm; Follow the tracks of the effluent liquid required for collecting with HPLC, satisfactory effluent liquid is merged, concentrates, desalted, is adjusted to pH=3.0 with 0.1mol/L hydrochloric acid, then concentrated, freeze-drying, obtains proteidin fine work.
From above-mentioned disclosed technical scheme; the present invention adopts RinkAmideMBHA resin to be starting raw material; HBTU/HOBt is condensing agent; the amino acid with Fmoc-blocking group is connected successively according to the method for solid phase synthesis; last amino acid uses Boc-Arg-OHHCl, obtains 17 peptide resins of protection, then adds and cuts peptide reagent and carry out cutting peptide; add ether sedimentation, obtain reduced form crude product.-SH base the Trt of 5-position and 14-position Cys protects, and is under the condition of 8.5 ~ 10.0 with hydrogen peroxide oxidation at pH;-SH base the Acm of 7-position and 12-position Cys protects, and with iodine oxidation in methanol solution, obtains two disulfide linkage Cys
5-14cys
7-12crude product, finally adopts HPLC(C8 post) carry out separation and purification, obtain target product, purity is 99.21%, and total recovery is 23.68%.Method of the present invention, production cost is low, and technique is simple, and environmental pollution is low, and productive rate is high.Method of the present invention is convenient to industrializing implementation, has larger industrialization prospect.
Embodiment
The materials list adopted in embodiment and aforementioned process is as follows:
| No | The name of an article | Production firm |
| 1 | Fmoc-Arg(Pbf)-OH（MW：648.8） | Sichuan Sangao Biochemical Co., Ltd |
| 2 | Fmoc-Gly-OH（MW:297.3） | Sichuan Sangao Biochemical Co., Ltd |
| 3 | Fmoc-Leu-OH（MW:353.4） | Sichuan Sangao Biochemical Co., Ltd |
| 4 | Fmoc-Cys(Trt)-OH（MW:585.7） | Sichuan Sangao Biochemical Co., Ltd |
| 5 | Fmoc-Cys(Acm)-OH(MW:414.5) | Sichuan Sangao Biochemical Co., Ltd |
| 6 | Fmoc-Tyr(tBu)-OH（MW:459.5） | Sichuan Sangao Biochemical Co., Ltd |
| 7 | Fmoc-Phe-OH（MW:387.4） | Sichuan Sangao Biochemical Co., Ltd |
| 8 | Fmoc-Val-OH（MW:339.4） | Sichuan Sangao Biochemical Co., Ltd |
| 9 | Boc-Arg·HCl·H 2O | Sichuan Sangao Biochemical Co., Ltd |
| 10 | Rink Amide mbha resin | Tianjin with become Science and Technology Ltd. |
| 11 | 1-hydroxy benzo triazole (HOBt) | Sichuan Sangao Biochemical Co., Ltd |
| 12 | TBTU(O-benzotriazole-N, N, N', N'-tetramethyl-urea a tetrafluoro borate) | Sichuan Sangao Biochemical Co., Ltd |
| 13 | DIC (N, N-DIC) | Suzhou Time-chem Technologies Co., Ltd |
| 14 | Trifluoroacetic acid (TFA) | The happy Industrial Co., Ltd. of Shanghai Jin Jin |
| 15 | Tri isopropyl silane (TIS) | Shanghai Bang Cheng Chemical Co., Ltd. |
| 16 | Dithioglycol (EDT) | Shanghai Jia Chen Chemical Co., Ltd. |
| 17 | N-methylmorpholine (NMM) | Shanghai Ke Fan Chemical Co., Ltd. |
| 18 | Dimethyl formamide (DMF) | The happy Industrial Co., Ltd. of Shanghai Jin Jin |
| 19 | Anhydrous methanol (MeOH) | Shanghai development chemical industry one factory |
| 20 | Hexahydropyridine (PIP) | The happy Industrial Co., Ltd. of Shanghai Jin Jin |
Embodiment 1
(1) preparation of Fmoc-Arg (Pbf)-resin
Take RinkAmideMBHA resin 200 grams (200 orders, 0.50mmol/g, 100mmol), soak with 2000mlDMF, make resin fully swelling, drain.Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add Fmoc-Arg (Pbf)-OH (MW:648.8 being dissolved in 1600mlDMF, 200mmol) 130g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(2) preparation of Fmoc-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Fmoc-Gly-OH (MW:297.3 being dissolved in 1600mlDMF, 200mmol) 59.5g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(3) preparation of Fmoc-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Fmoc-Val-OH (MW:339.4 being dissolved in 1600mlDMF, 200mmol) 67.9g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(4) preparation of Fmoc-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add Fmoc-Cys (Trt)-OH (MW:585.7 being dissolved in 1600mlDMF, 200mmol) 117.1g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(5) preparation of Fmoc-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Fmoc-Val-OH (MW:339.4 being dissolved in 1600mlDMF, 200mmol) 67.9g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(6) preparation of Fmoc-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add Fmoc-Cys (the Acm)-OH (414.5 being dissolved in 1600mlDMF, 200mmol) 82.9g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(7) preparation of Fmoc-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Fmoc-Phe-OH (387.4 being dissolved in 1600mlDMF, 200mmol) 77.5g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(8) preparation of Fmoc-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add Fmoc-Arg (Pbf)-OH (MW:648.8 being dissolved in 1600mlDMF, 200mmol) 130g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(9) preparation of Fmoc-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Fmoc-Gly-OH (MW:297.3 being dissolved in 1600mlDMF, 200mmol) 59.5g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(10) preparation of Fmoc-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add Fmoc-Arg (the Pbf)-OH(MW:648.8 being dissolved in 1600mlDMF, 200mmol) 130g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(11) preparation of Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add Fmoc-Cys (the Acm)-OH (414.5 being dissolved in 1600mlDMF, 200mmol) 82.9g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(12) preparation of Fmoc-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add Fmoc-Tyr (the tBu)-OH (459.5 being dissolved in 1600mlDMF, 200mmol) 91.9g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(13) preparation of Fmoc-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add Fmoc-Cys (Trt)-OH (MW:585.7 being dissolved in 1600mlDMF, 200mmol) 117.1g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 1 hour.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(14) preparation of Fmoc-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Fmoc-Leu-OH (353.4 being dissolved in 1600mlDMF, 200mmol) 70.7g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(15) preparation of Fmoc-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Fmoc-Gly-OH (MW:297.3 being dissolved in 1600mlDMF, 200mmol) 59.5g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(16) preparation of Fmoc-Gly-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Fmoc-Gly-OH (MW:297.3 being dissolved in 1600mlDMF, 200mmol) 59.5g, HBTU (MW:321,200mmol) 64.2g, HOBT (MW:153.1,200mmol) 30.6g, NMM50ml (MW=101.2), by mixture 22-24 DEG C of reaction 70 minutes.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
(17) preparation of Boc-Arg-Gly-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of 1600 milliliter of 23% hexahydropyridine, 22-24 DEG C is reacted 30 minutes.Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain.
Add the Boc-Arg-OHHClH being dissolved in 1600mlDMF
2o(MW:328.8,300mmol) 98.6g, DIC (MW:126.2,300mmol) 37.9g, HOBT (MW:153.1,300mmol) 45.9g, by mixture 20-22 DEG C of reaction 12 hours.Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain.
Three times are washed again with anhydrous methanol.After draining, put into vacuum drier dry, weigh, obtain protection 17 peptide resin 540g.Yield is: 98.26% ((540-200)/3460.19
protect 17 peptide molecular weights/ 100mmol
resin charging capacity=98.26%).
(18) H-Arg-Gly-Gly-Leu-Cys (H)-Tyr-Cys (Acm)-Arg-Gly-Arg-Phe-Cys (Acm)-Val-Cys (H)-Val-Gly-Arg-NH
2preparation
17 peptide resins of protection are transferred to and are cut in peptide bottle, under cooling, add while stirring and cut peptide reagent: (TFA/TIS/EDT/H
2o=3600ml/160ml/100ml/40ml), 15-18 DEG C of continuation stirring is carried out cutting reactive polypeptide 4.0 hours.Filter, drain, filtrate adds anhydrous diethyl ether precipitation.Obtain reduced form 17 peptide 195g crude product, yield is: 95.28%(195/2046.51
17 peptide molecular weights/ 100mmol
resin charging capacity=95.28%).
（19)
-NH
2single disulfide linkage Cys
5-14the preparation of crude product
Reduced form 17 peptide crude product is dissolved in (concentration is 2mg/ml) in purified water, under agitation slowly adds 1mol/L ammoniacal liquor, regulate PH to 8.5 ~ 10.0, add hydrogen peroxide 50ml and stir 30 minutes, follow the tracks of reaction process with HPLC, add acetic acid to pH=5 ~ 6, cross and filter insolubles.
（20）
Two disulfide linkage Cys
5-14cys
7-12the preparation of crude product
At single disulfide linkage Cys
5-14in the aqueous solution of crude product, under agitation slowly add the methanol solution (126g is dissolved in 1000 methyl alcohol) of iodine, stir about 60 minutes (HPLC follows the tracks of reaction process), drips 10% hypo solution termination reaction.After decompression removing methyl alcohol, reclaim iodine 4 times with carbon tetrachloride extraction.Use 0.4 μm of membrane filtration again.
(21) separation and purification
Filtrate through C8 post (10 μ, 200,15 × 25cm) separation and purification, moving phase: 0.16M phosphoric acid triethylamine: acetonitrile (75 ~ 85.5:25 ~ 14.5); Flow velocity is: 600ml/min; Determined wavelength is: 280nm; Follow the tracks of the effluent liquid required for collecting with HPLC, satisfactory effluent liquid is merged, concentrates, desalts, is adjusted to pH=3.0 with 0.1mol/L hydrochloric acid, then concentrated, freeze-drying, obtain 45g proteidin fine work, purity is 99.21%, and total recovery is: 23.68%(45g/1900.32
proteidin molecular weight/ 100mmol
resin charging capacity=23.68%).
<110> Shanghai Soho-yiming Pharmaceuticals Co., Ltd., Zhou Yiming, Cui Qi
The preparation method of a <120> synthesizing proteidin with solid phase polypeptide
<140>201110183315.0
<160>1
<210>1
<211>17
<212>PRT
<213> artificial sequence
<223> synthetic peptide
<400>1
ArgGlyGlyLeuCysTyrCysArgGlyArgPheCysValCysValGlyArg
151015
Claims (3)
1. a preparation method for synthesizing proteidin with solid phase polypeptide, comprises the steps:
With RinkAmideMBHA resin for starting raw material, HBTU/HOBt is condensing agent, the amino acid with Fmoc-blocking group is connected successively according to the method for solid phase synthesis, last amino acid uses Boc-Arg-OHHCl, obtain 17 peptide resins of protection, then add and cut peptide reagent and carry out cutting peptide, add ether sedimentation, obtain reduced form crude product;-SH base the Trt of 5-position and 14-position Cys protects, and is under the condition of 8.5 ~ 10.0 with hydrogen peroxide oxidation at pH;-SH base the Acm of 7-position and 12-position Cys protects, and with iodine oxidation in methanol solution, obtains two disulfide linkage Cys
5-14cys
7-12crude product, finally adopts high performance liquid phase C8 post to carry out separation and purification, obtains target product;
Said connection successively has the amino acid of Fmoc-blocking group, obtains protection 17 peptide resin, sloughs the method for Fmoc-blocking group therebetween successively, comprise the steps:
(1) preparation of Fmoc-Arg (Pbf)-resin
RinkAmideMBHA resin DMF soaks, and makes resin fully swelling, drains, and adds the DMF solution of hexahydropyridine, and 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain; Add the Fmoc-Arg (Pbf)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
Said RinkAmideMBHA resin substitution amount is: 0.3-0.6mmol/g;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(2) preparation of Fmoc-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Gly-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(3) preparation of Fmoc-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Val-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(4) preparation of Fmoc-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Cys (Trt)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(5) preparation of Fmoc-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Val-OH being dissolved in DMF, HBTU, HOBT, NMM, a mixture 22-24 DEG C reaction is connect peptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(6) preparation of Fmoc-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add be dissolved in DMF Fmoc-Cys (Acm)-OH, HBTU, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(7) preparation of Fmoc-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Phe-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(8) preparation of Fmoc-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Arg (Pbf)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(9) preparation of Fmoc-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Gly-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(10) preparation of Fmoc-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Arg (Pbf)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(11) preparation of Fmoc-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add be dissolved in DMF Fmoc-Cys (Acm)-OH, HBTU, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(12) preparation of Fmoc-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Tyr (tBu)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(13) preparation of Fmoc-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Cys (Trt)-OH, HBTU, HOBT, the NMM that are dissolved in DMF, mixture 22-24 DEG C is connect reactive polypeptide 1 hour; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(14) preparation of Fmoc-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add Fmoc-Leu-OH, HBTU of being dissolved in DMF, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(15) preparation of Fmoc-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Gly-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(16) preparation of Fmoc-Gly-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Fmoc-Gly-OH being dissolved in DMF, HBTU, HOBT, NMM, mixture 22-24 DEG C is connect reactive polypeptide 70 minutes; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(17) preparation of Boc-Arg-Gly-Gly-Leu-Cys (Trt)-Tyr (tBu)-Cys (Acm)-Arg (Pbf)-Gly-Arg (Pbf)-Phe-Cys (Acm)-Val-Cys (Trt)-Val-Gly-Arg (Pbf)-resin
Add the DMF solution of hexahydropyridine, 22-24 DEG C is reacted 30 minutes; Drain, respectively wash three times with DMF, anhydrous methanol, DMF respectively, drain;
Add the Boc-Arg-OHHClH being dissolved in DMF
2o, DIC, HOBT, connect reactive polypeptide 11 hours by mixture 20-22 DEG C; Drain, DMF, anhydrous methanol, DMF respectively wash three times, drain;
Three times are washed again with anhydrous methanol; After draining, put into vacuum drier dry, weigh, obtain protection 17 peptide resin;
The concentration of the DMF solution of said hexahydropyridine is: 22-24%;
The temperature of reaction of hexahydropyridine is 22-24 DEG C;
(18) H-Arg-Gly-Gly-Leu-Cys (H)-Tyr-Cys (Acm)-Arg-Gly-Arg-Phe-Cys (Acm)-Val-Cys (H)-Val-Gly-Arg-NH
2preparation
17 peptide resins of protection are transferred to and are cut in peptide bottle, under cooling, add while stirring and cut peptide reagent, carry out cutting reactive polypeptide 3.5-4.5 hour under 15-18 DEG C of stirring; Filter, drain, filtrate adds anhydrous diethyl ether precipitation, obtains reduced form 17 peptide crude product;
(19)
-NH
2single disulfide linkage Cys
5-14the preparation of crude product
Be dissolved in purified water by reduced form 17 peptide crude product, under agitation slowly add 1mol/L ammoniacal liquor, regulate pH to 8.5 ~ 10.0, add hydrogen peroxide 50ml and stir 30 minutes, HPLC follows the tracks of reaction process, adds acetic acid to pH=5 ~ 6, crosses and filters insolubles;
Crude product is dissolved in concentration in purified water: 1.5-2.5mg/ml;
By ammoniacal liquor adjustment pH scope be: 8.5 ~ 10.0;
(20)
two disulfide linkage Cys
5-14cys
7-12the preparation of crude product
At single disulfide linkage Cys
5-14in the aqueous solution of crude product, under agitation slowly add the methanol solution of iodine, stir about 60 minutes, HPLC follows the tracks of reaction process, drips 10% hypo solution termination reaction; After decompression removing methyl alcohol, reclaim iodine 4 times, with 0.4 μm of membrane filtration with carbon tetrachloride extraction;
The concentration of methanol solution of iodine is: 126g iodine is dissolved in 1000ml methyl alcohol;
(21) separation and purification
Filtrate is through the separation and purification of C8 filled column, and moving phase is: phosphoric acid triethylamine/acetonitrile; Flow velocity is: 500-600ml/min; Determined wavelength is: 260-280nm; Follow the tracks of the effluent liquid required for collecting with HPLC, satisfactory effluent liquid is merged, concentrates, desalted, is adjusted to pH=3.0 with 0.1mol/L hydrochloric acid, then concentrated, freeze-drying, obtain proteidin fine work, the English of described proteidin is called Iseganan.
2. method according to claim 1, is characterized in that, in step (18), said peptide reagent of cutting is: TFA/TIS/EDT/H
2o=3600ml/160ml/100ml/40ml; Temperature of reaction is: 15 ~ 18 DEG C; Reaction churning time is: 3.5 ~ 4.5 hours.
3. method according to claim 1, is characterized in that, in step (21), in said moving phase: the concentration of the phosphoric acid triethylamine aqueous solution is: 0.08 ~ 0.25mol/L; Phosphoric acid triethylamine aqueous solution proportion in acetonitrile is: 14.5 ~ 25.0; Determined wavelength is: 260-280nm; C8 filler is: 10 μ, 200.
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| Development of Protegrins for the Treatment and Prevention of Oral Mucositis: Structure–Activity Relationships of Synthetic Protegrin Analogues;Chen J.等;《Biopolymers》;20001231;第55卷;第96页右栏倒数第1段到第97页左栏 * |
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